A Chemically Modified Subfragment-L of Myosin from Skeletal Muscle As a Novel Tool for Identifying the Function of Actomyosin in Non-Muscle Cells1
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J. Biochem., 82, 105-115 (1977) A Chemically Modified Subfragment-l of Myosin from Skeletal Muscle as a Novel Tool for Identifying the Function of Actomyosin in Non-Muscle Cells1 Masahiro ISHIURA,2 Kazuko SHIBATA-SEKIYA,2 Toyoki KATO, and Yuji TONOMURA Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 560 Received for publication, January 29, 1977 Two kinds of subfragment-1 of myosin, S-1(T) and S-1(CT), were prepared by two-step tryptic [EC 3.4.21.4] digestion of myosin that had been modified with about 1 mol of p-chloromercuri benzoate (CMB) per mol of myosin, and one-step chymotryptic [EC 3.4.21. 1] digestion of the myosin, respectively. The amount of bound CMB was about 0.82-0.90 mol per 2 mol of S-1. Both kinds of S-1 modified with CMB equally inhibited superprecipitation of myosin B from rabbit skeletal muscle. About 2 mol of CMB-S-1 (1 mol of CMB-S-IA) inhibited the function of 1 mol of actin monomer on the superprecipitation of actomyosin reconstituted from myosin and fibrous actin(FA) with relaxing protein (RP). CMB-S-1 also effectively inhibited superprecipitation of myosin B from the plasmodia of the slime mold Physarum polycephalum. The ATPase [EC 3.6.1.3] activity of CMB-S-1(T) was similar to that of CMB-S-1(CT) in the absence of FA, but was not enhanced as effectively by FA as the latter. In the presence of 0.3 mg/ml of FA with RP, the activity of CMB-S-1(T) was only one-fifth of that of CMB-S-1 (CT). CMB-S-1(T) did not affect the activities of ATPase from animal cells outside actomyosin systems, such as the Ca 2+-dependent ATPase [EC 3.6.1.3] of the SR prepared from rabbit skeletal muscle and the Na+,K+-dependent ATPase [EC 3.6.1.3] from porcine kidney. It also scarcely affected Ca 2+-uptake by the SR at concentrations lower than 0.2 mg/ml. However, CMB-S-1(T) strongly inhibited the polymerization and depolymerization of tubulin prepared from bovine brain. At 0.15 mol per mol of tubulin heterodimer, CMB-S-1(T) 1 This investigation was supported by a grant for the Japan-U.S. Cooperative Science Program from the Japan Society for the Promotion of Science, and also by grants from the Ministry of Education, Science and Culture of Japan and the Muscular Dystrophy Association, Inc. 2 On leave from the Research Institute for Microbial Diseases, Osaka University, Yamada-Kami, Suita, Osaka 565. 3 Present address: Nayoro Women's College, Nayoro City, Hokkaido 096. Abbreviations: HMM, H-meromyosin; S-1, subfragment-1 of myosin; S-1(T), S-1 prepared by tryptic digestion of HMM; S-1(CT), S-1 prepared by chymotryptic digestion of myosin; S-1B, S-1 with the initial burst of Pi-libera tion; S-lA, S-i without the Pi-burst; FA, F-actin; RP, relaxing protein; SR, sarcoplasmic reticulum; CMB, p - chloromercuribenzoate; EGTA, ethyleneglycol his (ƒÀ-aminoethy]ether)-N,N•Œ-tetraacetate; Mes, 2-(N-morpholino) ethanesulfonic acid. Vol. 82, No. 1, 1977 105 106 M. ISHIURA, K. SHIBATA-SEKIYA , T. KATO, and Y. TONOMURA inhibited by 50% the extent of polymerization of 0.80 mg/ml tubulin (7.3 ƒÊm tubulin hetero dimer). S-l(T) also inhibited tubulin polymerization as effectively as CMB-S-1(T). CMB S-1(CMB-S-1A) also weakly bound itself to polymerized tubulin. It was concluded that CMB-S-1(T) can be used as a specific inhibitor of actin functions in non-muscle cells if the possible involvement of tubulin is excluded by other means. Actin, myosin, and tropomyosin have been isolated In the present paper, we describe experiments from many kinds of non-muscle cells and their showing that subfragment-l modified with CMB localization in cells has been studied (see review by (CMB-S-1) meets the requirement for an actin Pollard and Weihing, Ref. 1). Actin has been specific inhibitor. Previously, we showed that visualized in cells by selective decoration of actin CMB-S-1 is composed of equimolar S-lA modified filaments with HMM (2) or by specifically staining with CMB(CMB-S-lA) and S-1B not modified with them with fluorescent dye-labeled HMM (3). Re CMB, and that CMB-S-1 inhibits the superpre cently, antibodies specifically reactive with each cipitation of actomyosin induced by ATP, since component of contractile proteins from non-muscle CMB-S-1A binds very tightly with FA even in the cells, such as actin (4), myosin (5), tropomyosin (6), presence of ATP, and thus occupies all the myosin and ƒ¿-actinin (7), have been prepared and used binding sites of actin (26). In the present work, successfully to show the intracellular distribution we prepared two kinds of CMB-S-1, CMB-S-1(T), of each component. Much of the actin is localized and CMB-S-l(CT), and compared their inhibition on microfilaments (2, 4), and in some cells, myosin of the superprecipitation of actomyosin and their (5), tropomyosin (6), and ƒ¿-actinin (7) were also ATPase activities in the presence of FA. We associated with actin filaments. While no function found that the two kinds of CMB-S-1 inhibited has been established for these proteins, they have superprecipitation of actomyosin from rabbit been implicated in a variety of cell functions includ skeletal muscle with the same effectiveness, while ing motility, exocytosis, cytokinesis, membrane the acto-S-1-ATPase activity of CMB-S-i(T) was ruffling, maintenance of cell shape and cell adhesion much lower than that of CMB-S-l(CT). Thus, to substratum (8-13, see also Ref. 1 for review). using CMB-S-1(T), we studied the stoichiometric Cytochalasin B and D, microfilament-disrupt relation between CMB-S-1 and actin in the inhibi ing mold metabolites, have been widely used to tion of superprecipitation by CMB-S-1, and found identify the functions of microfilaments in non that 2 mol of CMB-S-1 (1 mol of CMB-S-lA) muscle cells (13-19). However, it remains difficult inhibited the function of 1 mol of actin monomer. to identify actin-specific functions in non-muscle To see the effectiveness of CMB-S-1 on actomyosin cells, since there is no direct evidence showing that of non-muscle origin, we prepared Physarum myosin cytochalasins specifically inhibit actin functions. B, and found that CMB-S-1 also effectively inhibited Cytochalasin D was reported to bind with myosin the superprecipitation of Physarum myosin B. To and inhibit myosin ATPase [EC 3. 6. 1. 3] (20). confirm the specificity of CMB-S-1, we prepared Cytochalasin B has the undesirable side effect of SR [EC 3.6.1.3] from rabbit skeletal muscle, Na+, inhibiting some cellular transport systems (21-24). K+-dependent ATPase [EC 3.6.1.3] from porcine To identify actin functions in non-muscle cells, kidney and tubulin from bovine brain, and studied first, we must find a substance which binds with their possible interactions with CMB-S-1. We actin specifically and stoichiometrically and inhibits found that they scarcely interacted with CMB-S-1 actin functions. Second, we must elaborate a except for tubulin. Therefore, we concluded that method of introducing the substance into cells CMB-S-1 can be used as a specific inhibitor of actin without damaging cell functions. One such method although it may also interact with tubulin. enables us to introduce any substance into a target cell by HVJ (Sendai virus)-mediated cell fusion EXPERIMENTAL between the target cell and a human erythrocyte ghost loaded with(25) the substance Materials-Myosin (MW=4.8 x 106) was pre J. Biochem. CMB-S-1 AS A SPECIFIC INHIBITOR OF ACTOMYOSIN 107 pared from rabbit skeletal white muscle by the NaOH (pH 6.5), at 4•Ž. Next, it was homo method of Perry (27). HMM (MW =3.4 x 105) genized in I ml of buffer per g of tissue with a was prepared by tryptic digestion of myosin by the Sorvall Omnimixer for I min at half-maximum method of Szent-Gyorgyi (28) with slight modifi speed. The homogenate was centrifuged at cation (29). S-i(T) (MW=1.2 x 105) was prepared 100,000 x q for 1 h at 4•Ž. The supernatant was by tryptic digestion of HMM, followed by chro collected, and mixed with an equal volume of the matography on Sephadex G-200, as described reassembly buffer containing 8 M glycerol, and previously (26, 30). S-1(CT) (MW=1.2 x 105) was incubated for 20 min at 37•Ž to polymerize tubulin. prepared by chymotryptic digestion of myosin at Polymerized tubulin was collected by 100,000 x g low ionic strength, as described by Weeds and centrifugation for I h at 25•Ž, then dissolved in Taylor (31), and purified on Sephadex G-200. cold reassembly buffer, which contained 1 mm GTP G-actin with RP was extracted from an acetone instead of ATP (GTP-reassembly buffer), by gentle powder of rabbit skeletal muscle at room tem homogenization in a glass homogenizer. Next, perature, and purified by a polymerization-depoly the mixture was chilled on ice for 30 min to de merization procedure (32). Purified G-actin was polymerize tubulin. The tubulin solution was prepared from an acetone powder of rabbit skeletal clarified by 100,000 x g centrifugation for 1 h at muscle by the method of Spudich and Watt (33).